丹紫康膝冲剂延缓膝骨关节炎模型大鼠软骨退变及软骨下骨异常骨重建的作用机制研究

Mechanism of Danzi Kangxi Granules on delaying cartilage degeneration and remodeling abnormal subchondral bone in Knee Osteoarthritis rats

目的探究丹紫康膝冲剂延缓膝骨关节炎(KOA)模型大鼠软骨退变及软骨下骨异常骨重建的可能作用机制。方法将40只SD雄性大鼠随机分为空白组、模型组、双氯芬酸钠组、丹紫康膝冲剂组,每组10只。空白组不做任何处理,其余3组采用Hulth法建立KOA模型。建模成功后,双氯芬酸钠组予5 mg/kg双氯芬酸钠灌胃,丹紫康膝冲剂组予1.67 g/kg丹紫康膝冲剂灌胃,空白组、模型组均予等容积0.9%氯化钠注射液灌胃,每日灌胃1次,连续28 d。采用苏木精-伊红(HE)染色和番红-O-固绿组织染色制作病理切片观察软骨病理损伤情况,并记录病理学退变指标;末端转移酶介导的DIG-dUPT切口末端标记法(TUNEL)观察软骨细胞凋亡情况;酶联免疫吸附法检测外周血白细胞介素1β(IL-1β)、IL-18、肿瘤坏死因子α(TNF-α)、Ⅱ型胶原C端肽(CTXⅡ)、Ⅱ型胶原C前肽(CPⅡ)水平;蛋白免疫印迹法(Western blot)检测B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)、基质金属蛋白酶3(MMP-3)、MMP-13、骨形态发生蛋白7(BMP-7)蛋白表达;取大鼠右侧胫骨近端关节离体标本进行Micro-CT扫描,测量软骨下骨微结构参数骨体积(BV);骨体积分数:骨体积/总体积(BV/TV)、骨小梁平均厚度(Tb.Th)、骨小梁数量(Tb.N)、体积骨密度(vBMD)。结果经HE染色,空白组大鼠软骨滑膜组织完整,细胞排列整齐,无炎症细胞浸润和基质损伤或蜕变。模型组大鼠软骨组织表面凸凹不平,软骨表层变薄,细胞排列不规则,此外可见大量炎症细胞、中性粒细胞、淋巴细胞浸润,而且深部软骨细胞可见明显细胞核压缩,膝关节宽度和关节退变深度显著增加。与模型组比较,丹紫康膝冲剂组大鼠软骨细胞排列均匀,胞核和胞质逐渐恢复正常。经番红-O-固绿染色,空白组大鼠软骨组织染色均匀,结构清晰。模型组大鼠骨关节面不平,软骨钙化,胶原纤维增生,软骨细胞外基质经番红O染色变浅,染色不均一,而且软骨陷窝结构扩张。与模型组比较,丹紫康膝冲剂组大鼠软骨组织番红O稍淡染,潮线逐渐清晰,细胞排列逐渐均匀。与空白组比较,模型组大鼠关节软骨基质丢失宽度、改良的国际骨关节炎研究会(OARSI)评分、软骨退变总宽度、软骨退变最显著处的宽度均增加(P<0.05);与模型组比较,双氯芬酸钠组、丹紫康膝冲剂组大鼠上述指标则减少(P<0.05);双氯芬酸钠组与丹紫康膝冲剂组大鼠上述指标比较差异无统计学意义(P>0.05)。与空白组比较,模型组大鼠软骨细胞凋亡指数升高(P<0.05);与模型组比较,双氯芬酸钠组、丹紫康膝冲剂组大鼠软骨细胞凋亡指数降低(P<0.05);与双氯芬酸钠组比较,丹紫康膝冲剂组软骨细胞凋亡指数降低(P<0.05)。与空白组比较,模型组大鼠血清IL-1β、IL-18、TNF-α、CTXⅡ水平均升高,CPⅡ水平降低(P<0.05);与模型组比较,双氯芬酸钠组、丹紫康膝冲剂组大鼠血清IL-1β、IL-18、TNF-α、CTXⅡ水平均降低(P<0.05),CPⅡ水平升高(P<0.05);与双氯芬酸钠组比较,丹紫康膝冲剂组大鼠血清IL-1β、IL-18、TNF-α、CPⅡ水平均升高(P<0.05),CTXⅡ水平降低(P<0.05)。经Western blot检测,与空白组比较,模型组大鼠软骨组织Bax、MMP-3、MMP-13蛋白表达量升高(P<0.05),Bcl-2、BMP-7蛋白表达量降低(P<0.05);与模型组比较,双氯芬酸钠组、丹紫康膝冲剂组大鼠软骨组织Bax、MMP-3、MMP-13蛋白表达量均降低(P<0.05),Bcl-2、BMP-7蛋白表达量均升高(P<0.05);与双氯芬酸钠组比较,丹紫康膝冲剂组大鼠软骨组织Bcl-2、Bax、MMP-3蛋白表达量降低(P<0.05),BMP-7蛋白表达量升高(P<0.05)。经Micro-CT扫描,与空白组比较,模型组大鼠胫骨BV/TV、Tb.Th、Tb.N、vBMD均降低(P<0.05);与模型组比较,双氯芬酸钠组、丹紫康膝冲剂组大鼠胫骨BV/TV、Tb.Th、Tb.N、vBMD均升高(P<0.05);与双氯芬酸钠组比较,丹紫康膝冲剂组大鼠胫骨BV/TV、Tb.Th、Tb.N、vBMD均升高(P<0.05)。模型组大鼠胫骨近端软骨表层可见大量基质迫损或丢失,软骨下骨骨小梁数量减少、排列紊乱并出现断裂现象。与模型组比较,丹紫康膝冲剂组大鼠软骨表层基质虽然也存在破损,但是最深至浅层或中层,尚未累及软骨下骨;软骨下骨骨小梁排列整齐,关节边缘未见骨赘形成。结论丹紫康膝冲剂可有效降低KOA模型大鼠炎症细胞因子分泌和MMPs表达,减轻软骨组织病理性损伤,改善KOA早期软骨下骨生物力学性能和微结构改变,从而延缓KOA大鼠模型软骨组织进行性退变和软骨下骨异常骨重塑的发生。

Objective To discuss the effect of Danzi Kangxi Granules on delaying cartilage degeneration and remodeling abnormal subchondral bone in Knee Osteoarthritis(KOA)rats.Methods Forty SD rats were randomly grouped,10 rats in each.KOA model was established by Hull method except for Blank group.The gastric irrigation were performed on the four groups,which Blank group and Model group(0.9%sodium chloride injection),Diclofenac Sodium group(5 mg/kg),Danzi Kangxi Granules group(1.67 mg/kg)after successful,once daily for 28 days.Pathological sections were perfromed to observe the pathological damage of cartilage by hematoxylin-eosin(HE)staining and safranin-O-fast green tissue staining andpathological degenerationindicators were recorded.Terminal transferase-mediated DIG-dUPT cut end labeling Method(TUNEL)to observe the apoptosis of chondrocytes;ELISA.Enzyme-linked immunosorbent assay(ELISA)was to detect serum interleukin(IL)-1β,IL-18,tumor necrosis factorα(TNF-α),collagen typeⅡCtelopeptide(CTXⅡ),typeⅡpro-collagen C-propeptide(CPⅡ)levels.The apoptosis of chondrocytes was observed by TUNEL fluorescence staining.Western blot detection of B lymphoma-2 gene(Bcl-2),Bcl-2 related X protein(Bax),matrix metalloproteinase 3(MMP-3),MMP-13,bone morphogenetic protein 7(BMP-7)protein expression;Take the isolated specimen of the proximal joint of the right tibia of the rat for Micro-CT scan to measure the subchondral bone microstructure parameter bone volume(BV);bone volume fraction:bone volume/total volume(BV/TV),average trabecular bone thickness(Tb.Th),number of trabecular bone(Tb.N),volume bone density(vBMD).Results After HE staining,the cartilage and synovial tissue of the blank group was intact,the cells were arranged neatly,and there was no inflammatory cell infiltration and matrix damage or degeneration.In the model group,the cartilage tissue surface is uneven,the cartilage surface layer is thinned,and the cell arrangement is irregular.In addition,a large number of inflammatory cells,neutrophils,and lymphocytes can be seen infiltration,and deep chondrocytes can be seen with obvious nucleus compression,knee joint width and joint regression The depth of change increases significantly.Compared with the model group,the chondrocytes in the Danzi Kangxi Granules group were evenly arranged,and the nucleus and cytoplasm gradually returned to normal.After safranin-O-fast green staining,the cartilage tissue of the blank group was evenly stained and the structure was clear.In the model group,the bone articular surface is uneven,cartilage calcification,collagen fiber hyperplasia,cartilage extracellular matrix is lightened by safranin O staining,the staining is uneven,and the cartilage lacuna structure is expanded.Compared with the model group,the cartilage tissue of rats in the Danzi Kangxi Granules group was slightly stained with Safranin O,the tide lines were gradually clear,and the cell arrangement was gradually uniform.Compared with the blank group,the width of articular cartilage matrix loss,OARSI score,total width of cartilage degeneration,and the most significant width of cartilage degeneration in the model group increased(P<0.05);compared with the model group,the diclofenac sodium group,Dan The above indexes of rats in the Zikangxi granule group decreased(P<0.05);there was no significant difference in the above indexes between the diclofenac sodium group and the Danzi Kangxi Granules group(P>0.05).Compared with the blank group,the chondrocyte apoptosis index in the model group increased(P<0.05);compared with the model group,the chondrocyte apoptosis index in the diclofenac sodium group and the Danzi Kangxi Granules group decreased(P<0.05);Compared with the diclofenac sodium group,the chondrocyte apoptosis index in the Danzi Kangxi Granules group was lower(P<0.05).Compared with the blank group,the serum levels of IL-1β,IL-18,TNF-α,CTXⅡin the model group increased,and the CPⅡlevel decreased(P<0.05);compared with the model group,the diclofenac sodium group,Danzi Kangxi Granules group serum IL-1β,IL-18,TNF-α,CTXⅡlevels of rats in the granule group decreased(P<0.05),and the level of CPⅡincreased(P<0.05);compared with the diclofenac sodium group,the Danzi Kangxi Granules group was larger The levels of serum IL-1β,IL-18,TNF-αand CPⅡin mice increased(P<0.05),and the level of CTXⅡdecreased(P<0.05).By Western blot detection,compared with the blank group,the protein expression of Bax,MMP-3 and MMP-13 in the cartilage tissue of the model group increased(P<0.05),and the protein expression of Bcl-2 and BMP-7 decreased(P<0.05);Compared with the model group,the expressions of Bax,MMP-3,and MMP-13 proteins in the cartilage tissue of rats in the diclofenac sodium group and the Danzi Kangxi Granules group were reduced(P<0.05),and the Bcl-2 and BMP-7 proteins The expression levels were all increased(P<0.05);compared with the diclofenac sodium group,the Bcl-2,Bax,and MMP-3 protein expressions in the cartilage tissue of the Danzi Kangxi Granules group decreased(P<0.05),and the BMP-7 protein The expression level increased(P<0.05).Compared with the blank group,the tibia BV/TV,Tb.Th,Tb.N and vBMD of the model group were all decreased(P<0.05);compared with the model group,the tibia BV of the rats in the diclofenac sodium group and the Danzi Kangxi Granules group BV/TV,Tb.Th,Tb.N and vBMD were all increased(P<0.05);compared with the diclofenac sodium group,the tibia BV/TV,Tb.Th,Tb.N and vBMD of rats in the Danzi Kangxi Granules group were all increased Increase(P<0.05).After Micro-CT scan,the surface layer of the proximal tibia of rats in the model group showed a large amount of matrix compression or loss,the number of subchondral bone trabecula was reduced,the arrangement was disordered,and the phenomenon of fracture occurred.Compared with the model group,although the cartilage surface matrix of the rats in the Danzi Kangxi Granules group is also damaged,it is deepest to the superficial or middle layer and has not involved the subchondral bone;the subchondral bone trabeculae are arranged neatly,and no osteophytes are seen on the joint edges form.Conclusion Danzi Kangxi Granules can effectively reduce the secretion of inflammatory cytokines and the expression of MMPs in KOA model rats,reduce the pathological damage of cartilage tissue,improve the biomechanical properties and microstructure changes of subchondral bone in the early stage of KOA,thereby delaying the progress of cartilage tissue in KOA rat model Degeneration and abnormal bone remodeling of subchondral bone.